Project Summary Understanding how the CD4 T cells that harbor HIV in vivo differ from those that do not may spur the development of prophylactic or therapeutic antiviral strategies. Unfortunately, due to the complexity of HIV-infected CD4 T cell populations and to key technical obstacles, the detection and manipulation of these rare cells in blood and lymphoid tissue samples from HIV-positive individuals has previously been infeasible. We have therefore developed a novel technology termed PCR-activated cell sorting (PACS) that allows identification and manipulation of individual CD4 T cells in microfluidically-generated water-in-oil emulsions by the presence of intracellular HIV genomic DNA. In the proposed investigations, we will use this technology to sort single HIV-infected CD4 T cells from blood and lymphoid tissue of HIV-positive individuals for gene expression profiling by whole transcriptome deep sequencing. This comprehensive approach will allow discovery of novel markers of HIV-infected CD4 T cell in vivo, which will then be validated in follow-up tissue staining and cell sorting studies. Importantly, access to well-characterized NIH Clinical Center cohorts of HIV- positive study participants with spontaneous control of the virus, with uncontrolled infection, and with virus suppression on antiretroviral therapy (ART) will allow targeted study of these distinct clinical subgroups. Comparing results from these subgroups will allow differences between HIV infected CD4 T cells under differing degrees of natural or treatment-induced virologic suppression to be understood. By thus elucidating the determinants of HIV target cell selection and persistence in vivo, these studies may lead to the development of novel HIV cure strategies.